Hemoglobin A1c (HbA1c) and glycoalbumin that are glycosylated proteins included in the blood are often measured as a glycemic control marker for diabetics. These glycosylated proteins are produced by nonenzymatic reaction of D-glucose included in the blood with an amino-acid residue that constitutes a blood protein. Therefore, these glycosylated proteins clearly reflect glucose quantity included in the blood.
Main glycosylated sites of the protein included in the blood are ε-amino group of an internal lysine residue and α-amino group of an amino terminal amino acid (N terminal amino acid). For example, the HbA1c is a glycosylated protein that is generated as a result of binding of D-glucose to α-amino group of valine which is the N terminal amino acid of a hemoglobin β chain.
Recently, there has been developed an enzymatic measurement method (hereinafter referred to as an “enzyme method”) that measures the glycosylated protein included in the blood easily and in a short time. The enzyme method has been already commercialized. The use of the enzyme method allows high-throughput measurement of the glycosylated protein. The enzyme method is useful in the clinical examination field.
The enzyme method is conducted as follows. First, a protease hydrolyzes a glycosylated protein into glycosylated amino acids such as fructosyl valine, fructosyl lysine and fructosyl valyl histidine. Subsequently, such glycosylated amino acids are oxidatively hydrolyzed by use of fructosyl amino acid oxidase (FAOD). Thereafter, hydrogen peroxide generated by the oxidase reaction is colorimetrically determined by peroxidase chromogenic reaction system (see Patent Literatures 1 through 11).
In a case where the glycosylated protein is measured by the enzyme method, a substrate specificity of the fructosyl amino acid oxidase that is a main reaction enzyme is an important factor. For example, in a case where the HbA1c is measured, it is preferable to use an enzyme that has an excellent substrate specificity to fructosyl valine.
Further, in order to measure specifically to a β chain of the glycosylated hemoglobin, it is preferable to use an enzyme that acts on fructosyl valyl histidine. This is because N terminal amino acids of an α chain and a β chain of hemoglobin are both valines, and it is therefore necessary to recognize two amino acid residues at N terminal (that is, fructosyl valyl histidine) in order to measure specifically to the β chain (see Patent Literatures 12 and 13).
A bacteria that produces an oxidase that acts on fructosyl valyl histidine has been screened so far (see, for example, Patent Literature 14).
It has been reported to use genetically modified bacteria in order to produce, extract and purify the oxidase that acts on fructosyl valyl histidine. It has been also reported isolating a gene that encodes for the oxidase (see Patent Literatures 15 and 17, and Non-Patent Literatures 1 and 2).